1. Field of the Invention
The present invention relates to strains and methods for simple and efficient extraction of biological material from bacteria. The use of Escherichia coli (E. coli) and other commonly used microorganisms in research laboratories depends on the ability to prepare lysates to isolate the desired products under investigation. The present invention includes engineered E. coli strains that are inducible for rapid controlled lysis referred herein to as “autolysis”. The XJa strain was made from parental JM109 or BL21 strains by insertion of a chromosomally-encoded lytic gene to replace the tightly regulated araB gene. Thus, arabinose becomes a non-metabolizable inducer that induces the controlled autolysis phenotype. Upon induction of the λ R endolysin the E. coli remain physically intact but are efficiently lysed after one freeze-thaw cycle. The present invention is usable with many different buffer systems and is flexible in this regard. The controlled autolysis phenotype shows increased yields and purity of extracted protein compared to traditional lysozyme digestion, detergent based buffers, the harsh sonication, or French press mediated lysis methods. The present invention is useful for routine protein expression, isolation of nucleic acids, or other cellular material. In addition it is useful for high-through-put manipulation involving extraction of such material from E. coli., related bacteria, and other microorganisms known and commonly used in the art
2. Description of the Related Art
The need for simple and efficient extraction of material from bacteria such as E. coli, and other microorganisms is an ongoing concern for research scientists. Advances in the study of recombinant proteins had led to numerous methods together with variations to address common problems associated with obtaining complete lysis of the samples. Traditional protocols utilize purified preparations of lysozyme to generate spheroplasts and this method is still in common usage. Use of lysozyme has problems in that commercial preparations are often contaminated to some degree with lytic as well as other enzymes that can prematurely lyse the cells, degrade proteins, or nucleic acids. Also the lysozyme is an approximately 15 kDa protein that can mask or interfere with or mask proteins that are being expressed. Modified methods to solve these problems include using reduced concentrations of lysozyme (Marvin and Witholt 1987), osmotic shock, treatment with chloroform (Ames et al. 1984), and use of polymixin B, especially for release of periplasmic proteins (Cerny and Tueber 1971). However, they are inconsistent and do not completely solve the limitation of this method.
The use of a French press to lyse bacterial cultures is useful for large volume samples, but the limited availability of such equipment and high cost are often prohibitive. In addition the use of a French press is time consuming and not well suited for small samples or high throughput sample processing (Goeddel et al 1979, Schumacher et al. 1986). Use of sonication for bacterial lysis is common and ensures thorough lysis, but has troubles of increased sample handling time and heat generation both of which can be problematic, especially for screening purposes. Both the French press and sonication methods mediate extremely severe lysis that further disrupts and releases membrane proteins that can interfere with the purification of target proteins. The method of successive freeze thaw cycles is both time consuming and inefficient. This method often leads to degradation and also suffers from populations of unlysed or incompletely lysed cels. The use of detergent and salt containing buffers to mediate lysis, some of which are commercially available, are somewhat convenient, but suffer in that they introduce of materials that later can be problematic in enzymatic or functional assays (Novagen Corp). In addition they are not effective at extracting large molecular weight proteins.
The bacteriophage T7 lysozyme commercially available and encoded by the plasmid variants pLyseS, or pLyseE can be used to facilitate lysis, since the T7 enzyme can degrade the peptidoglycan cell wall. However, functionally strains carrying these genes are extremely fragile due to basal level expression. Also the system sometimes does not produce significant amounts of recombinant protein (Studier, 1990, Novagen Corp). The T7 lysozyme also inhibits T7 RNA polymerase and so is intended to reduce basal or leaky expression of potentially burdensome proteins until induction of the recombinant protein is desired. In addition the lytic enzymes of other well studied bacteriophages, or organisms could also be used, but have not been developed adequately for efficient lysis of E. coli or other bacteria for primary use in recombinant protein production methods to date (Young et al. 1992).
Thus there exists a strong need for improved methods of bacterial lysis to facilitate high-through put screening and also to reduce cost and sample handling time. The first step in obtaining material such as recombinant protein or nucleic acids from bacterial cells is to efficiently break them open, or lyse them. This fundamental process continues to be problematic in part due to the bacterial cell wall. There exists a need of alternative improved methods. The ability to rapidly and gently lyse bacteria would additionally enhance protein purification high-through put analysis and would also result in significant savings of time and money.